大花細辛授粉生物學及種子傳播機制

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(1)國立臺灣師範大學生命科學系碩士論文. 大花細辛授粉生物學及種子傳播機制 Pollination Biology and Dispersal Mechanism of Asarum macranthum Hook. f.. 研究生：莊貴竣 Kuei-Chun Chuang. 指導教授：王震哲博士 Jenn-Che Wang. 中華民國一百年一月.

(2) Acknowledgment 曾經有人問我，如果以 1~10 分來評比你研究生涯的順利度。我還記得我的回答是 8 分。在碩士班的這段時間，雖然遇到了非常多的困難與無數次的挫敗，但幸運的是，總是有許多重要的人在我最需要幫助的時候出現在我的生命當中。首先最感謝的就是王震哲老師，從大學就在老師的門下學習，從大學的專題到現在的碩士論文，感謝老師對我這個與實驗室研究主軸大不相同的研究的支持及肯定，在與老師的多次討論之下，使我對於論文的內容以及架構更加透徹，老師在進度報告上每一句對我肯定的話語，都成為在我研究上大力推進的動力，老師雖然有時嘴巴不說，但對學生的關心我都了然於心。再來要感謝王俊能老師，在二格山上與王俊能老師的巧遇，在與老師的討論後，更加深我對於研究的熱情，並且在台大與壁報展中與老師的一番對談，老師對我未來規畫的關心，更使學生心頭一陣暖意浮起。再來更要十分感謝蘇夢淮老師在論文上一字一句詳細的建議與指教，我永遠也忘不了口詴後老師給我的那兩張滿滿用電腦打字充滿寶貴意見的 A4 紙。在大花細辛這個題目之前，其實還有著一個蘭花的題目，而在研究蘭花授粉的路途上，植物界的前輩與先進們對小弟我的提攜，更是讓小弟難以忘懷。余大哥無私的開車載我出去認識這些美麗的蘭花，並教導我對待這些植物該有的態度，智凱哥哥在多本重要書籍的慷慨提供及言語上的支持與鼓勵，天銓在電話中寶貴地點的提供，雖然這個題目之後無法繼續下去，但你們對我而言是莫大的支持。而在大花細辛的研究當中，從大三一路走到現在，近五年的時間中，首先要感謝的便是長澤學長及淑娟學姐，長澤學長對於這個題目的啟蒙，以及適時地且默默地提供我許多重要的意見，以及文獻上的討論，甚至開車載我出門時，我兩秒就睡著及醒來就大吵大鬧的容忍；再來是淑娟學姐，一剛開始進入實驗室，因為有了學姐，我才能更認識並更融入實驗室的軌道當中，每次在我報告之前，總是學姐默默的在我身邊聽我報告且給予我很多一槍斃命的意見，再加上後期大約七百多次的論文修改，更令我感動的是，學姐常常都跟其他的人大力肯定我在實驗上的努力，小弟我都點滴在心頭，在此十分感謝兩位對小弟我的照顧。在研究上，感謝徐鈺鸚老師對於統計方法上所給予的指導，讓我的研究更增添了許多數據上顯著的支持；感謝胡愛群老師在研討會上與我討論彼此授粉研究上的新發現及出現的問題；謝秀梅老師實驗室螢光顯微鏡設施的借用以及共軛焦顯微鏡的協助操作；感謝吳忠信老師實驗室 DV 的借用，讓我的實驗更加事半功倍；感謝趙偉村老師對於空間分析上提供的意見與想法；以及萬分感謝師大化學系沈英宏學長對於氣味分析的全力支持，以及 GC-MS 操作的協助，還有對我無數氣味分析問題的解答。再來，實驗室的成員，我愛你們，感謝大家在每次的進度報告總是將我問到體無完膚，不過也是因為你們的寶貴問題，更是讓我對於論文能有更進一步的檢視其不足的空間，珆碩學長對於植物學識上的教導以及單眼相機的勸敗，甚至是無私的整個防潮箱攝影器材的借用，都讓我的實驗更加便利；明哲學長，感謝你身為授粉界前輩所給予我的寶貴意見，雖然有時你的笑聲會震破我的耳膜，不過有了你，讓我在實驗設計上注意到許多我以前忽略掉的細節；智凱學長每次看到我給與我肩膀上重重的輕拍以及鼓勵，著實能讓我懷著滿滿的動力及斷裂的鎖骨繼續往前；福隆學長在討論時總是有許多非常天馬行空的想法，這也提供了我在論文的討論上一個很大發揮的意見來源；杏倩及依恆對於實驗經費報帳上的協助，讓我在做實驗上無後顧之憂；威廷在先前蘭花.

(3) 研究上對於繩索攀樹技巧的協助及建議以及細辛研究時的寶貴意見；大胖在登山時總是協助我檢視裝備的完備及背包的填充，以及流行音樂的補給還有美味食物的提供；而我碩士班的好同學們，感謝你們在碩士班的一路相伴，親愛的李大王，雖然我這輩子都不可能跟你有任何更進一步的關係(因為你高攀不上)，不過你真的是我碩士班當中非常重要的人物，因為你讓我的生活更加的豐富，與你一起當孬種的日子真的好氣又好笑；承瑞同學總是能用犀利的字眼找出我研究上的漏洞及包容我凡事不求甚解的個性，與你們一同修課、一同研究、最後能一同畢業這都是我的榮幸。實驗室的學弟妹們，小聿、元馨、懿洲，實驗室因為你們的加入出現了嶄新的面貌，希望你們都能夠在研究的路途上和我一樣遇到這麼多的好人及貴人相助；也感謝小聿在我這學期與我一起擔任種分實驗助教時，協助我分擔採集的事務，因為有你，讓我在研究與教學上都能夠兼顧，此外，更要感謝實驗課旁聽的王媽媽對我的肯定與鼓勵。感謝小天多次協助實驗觀察的進行以及妙楓協助我與廣大師大粉絲之間的溝通及聯繫。另外，實驗室已經畢業的學長姐，你們對小弟我的照顧我真的沒齒難忘，感謝福麟、小量、奇異筆、小胖、惠如、家進，多謝你們對於我的支持與鼓勵。在二格山樣區與我渡個無數個日夜的夥伴們，我要敬上我最高的敬意，沒有你們，這些相當寶貴的授粉影片不會存在。感謝二格山上每天 3:00 固定聚會的大哥大姐們，總是提供我最溫暖的問候及不斷問我何時要畢業的話語，還有包容我不小心在步道上睡著時，繞過我而不往我的臉上踩下去的貼心；感謝小蛇、家進、巴布、大胖，在之前香蘭的樣區的開車相助，沒有你們，我這輩子都無法到達我的樣區；感謝小天，你與我度過了在二格山上的多數夜晚，與你一起仰望星空及在涼亭熱舞真是在山上消磨時間的最好活動；感謝李大王及二樓朋友，在山上掏心掏肺，幾度哭到無法自己，也讓我更加能夠面對過去，大講別人壞話時，更能抒發心中壓力；感謝珆碩學長，半夜一點的麥當勞快遞，讓我在二格山上感受到無比的溫馨及油炸食物的魅力；感謝承瑞，在二格山上有怪聲出現時，我只顧自己睡覺而棄你於不顧時的堅強；感謝蔣怡，在香蘭樣區時的陪伴以及突然暴雨時我倆共同在高漲的溪水中搶救 DV 的毅力；感謝斌垚，在香蘭樣區時雖然你一直認植物而忘了煮飯，但我還是相當感謝你的陪伴，讓這趟旅程增加了許多有趣的故事；感謝在二格山上曾經把我的 DV 拿走的民眾，因為有你，我變得更加堅強，更加能夠承受打擊。最後是我的好友們，可萱、詵雯、天駿及國修，我已經忘記我是第幾百次跟你們在聚會上分享與抱怨我的大小事，因為有你們靜靜的聆聽，我的情緒才能夠得到適當的宣洩，也因為每次與你們再一起，彷彿什麼事才都能夠一笑置之，有你們真的好棒；特別感謝可萱，雖然你一直用各種巧妙的理由迴避去二格山的邀約，不過論文英文的修正以及你看 5 頁才開始不耐煩的好脾氣對我而言真是意義重大，不定時電話的情緒宣洩更是我相當仰賴的管道；此外在香蘭研究時，感謝可萱陪伴我在晚上 3 點時討論原子說及帝王斑蝶來消弭時間；以及在樣區夜晚錄青蛙聲音的詵雯，抱歉~我睡著了。在授粉研究的路上一路走來，雖然孤單，但是因為有你們的支持，我能更加昂首闊步的走出我的下一步，沒有你們，我很難繼續支持到現在。現在若讓我重新評分~我要給我研究的順利度 10 分，因為我真的是太幸運了，能擁有你們~我愛你們~.

(4) Contents 中文摘要 ................................................................................................................................................ IV ABSTRACT ........................................................................................................................................... VI. GENERAL INTRODUCTION ................................................................................................................ 1. Part1- Pollination Biology of Asarum macranthum INTRODUCTION .................................................................................................................................... 3 MATERIAL AND METHODS ................................................................................................................ 6 RESULTS ............................................................................................................................................... 13 DISCUSSION ........................................................................................................................................ 30 REFERENCES ....................................................................................................................................... 36 APPENDIX-ANILINE BLUE STAINING ............................................................................................ 39. Part2- Dispersal Mechanism of Asarum macranthum INTRODUCTION .................................................................................................................................. 41 MATERIAL AND METHODS .............................................................................................................. 43 RESULTS ............................................................................................................................................... 45 DISCUSSION ........................................................................................................................................ 53 REFERENCES ....................................................................................................................................... 57. I.

(5) Figure Contents Figure 1– Habitat and morphology of Asarum macranthum __________________________________ 7 Figure 2– Three separate areas of dissected calyx tube _____________________________________ 11 Figure 3– Osmophore evaluation zone __________________________________________________ 11 Figure 4- Reproductive table of Asarum macranthum ______________________________________ 15 Figure 5– Arrangement for two whorls of anthers _________________________________________ 16 Figure 6– Pistil receptivity test – aniline blue staining ______________________________________ 16 Figure 7- Legitimate pollinators of Asarum macranthum ___________________________________ 17 Figure 8- Isopod- images of scanning electronic microscope ________________________________ 19 Figure 9-Fungus gnat- images of scanning electronic microscope ____________________________ 19 Figure 10- Video images of the pollinator “Isopods” _______________________________________ 20 Figure 11- Video images in night vision of the pollinator “isopods” ___________________________ 20 Figure 12- Video images of the pollinator “fungus gnat” ____________________________________ 21 Figure 13- Eggs of fungus gnat laid in the calyx tube ______________________________________ 24 Figure 14- Fisher‟s exact test – ovipositing and fruiting success correlation ____________________ 24 Figure 15- Chi-square test of egg-laying area _____________________________________________ 24 Figure 16- Behavior of isopods inside calyx tube __________________________________________ 25 Figure 17- The scent glands location of the Asarum macranthum _____________________________ 26 Figure 18- Scent chromatogram of Asarum macranthum and blank air ________________________ 27 Figure 19- Scent chromatograms in 7.861min ____________________________________________ 28 Figure 20- New volatile chromatogram after eliminating background interferences ______________ 29 Figure 21- Volatile chromatogram of oleic acid (modified from the figure in Christie, 2005) _______ 29 Figure 22- The dispersal agent composition of Asarum macranthum __________________________ 46 Figure 23- Behavior of myrmecochory __________________________________________________ 47 Figure 24- Ant dispersal curve of distance _______________________________________________ 49 Figure 25- Dispersal distance of three main ant species _____________________________________ 50 Figure 26- Removal ratio of “loss of function experiment” __________________________________ 51 Figure 27- Distribution pattern analysis _________________________________________________ 52. II.

(6) Table Contents Table 1–Pollination manipulations of Asarum macranthum _________________________________ 13 Table 2- Number and pollen load on visitors caught in the calyx tube _________________________ 18 Table 3- Visiting frequency and time (recorded by digital video recorder) ______________________ 18. III.

(7) 中文摘要在植物的繁殖過程當中，授粉及種子傳播為相當關鍵的兩項步驟。植物長期和生物媒介共演化的結果也使得這兩項步驟衍伸出許多精巧且多樣的設計，但由於缺乏研究也使得這些機制未明且充滿不確定性。因此，對於授粉系統與傳播模式詳盡且深入的研究有助於了解與記錄植物的族群動態以及繁殖策略的成功與否，更進一步地了解植物與媒介共演化後的巧妙機制。細辛屬植物因為花果皆位於地表，特殊的習性使得其生殖生物學充滿臆測，再加上族群大多呈現小族群群集分布的模式，使得授粉及種子傳播這兩項步驟倍受矚目。故我們針對授粉及種子傳播來進行研究以釐清細辛屬植物的繁殖機制。植物常利用擬態的方式來吸引授粉媒介前往拜訪，特化的花部構造模擬食物資源、配偶以及產卵場所。Vogel 於 1978 年對於細辛屬植物的授粉提出一項假說，認為細辛萼筒內壁具有許多網格狀的稜脊，其形態與擔子菌綱的蕈褶相仿，故可吸引原來在蕈褶內產卵的蕈蠅轉移產卵場所，藉模擬蕈褶吸引蕈蠅將卵產在細辛萼筒內部，在進出產卵的過程中協助授粉。但有關此授粉機制的假說迄今仍缺乏有統計效力的數據予以驗證，故在本研究中，觀察並記錄台灣特有種大花細辛的生殖生態以驗證 Vogel‟s hypothesis，並進一步以統計方式呈現蕈蠅產卵現象對成功授粉的影響，且於台灣北部低海拔山區設立一樣區進行長期觀察。根據自然及人工授粉結實率、花型變化、雌雄蕊活性檢測的研究結果顯示大花細辛為一雌先熟(protogynous)且自交親合(self-compatible)的物種，但雄蕊不會自行運動，因此不具自力授粉機制，必頇藉由媒介來完成授粉。本研究觀察到蕈蠅會於細辛萼筒內部的網格中產卵，支持 Vogel 於 1978 年所提出的授粉假說，顯示蕈蠅是大花細辛的重要傳粉媒介。Fisher‟s exact test 也證實產卵行為與結實率之間具有顯著相關。氣味分析的結果也佐證了大花細辛與蕈類的氣味相仿，皆具有類似油酸的氣味釋出，更加支持大花細辛模擬蕈蠅生育環境的授粉假說。此外，本研究也首次觀察到鼠婦傳粉的現象，由於細辛花開在接近地表處，. IV.

(8) 其陰暗且潮詴的萼筒內部形成了良好的庇護所，鼠婦躲藏及進出的行為也完成授粉。兩個授粉系統並存之下使得大花細辛的野外族群具有約五成的結實率。在種子傳播機制方面，本研究確認了大花細辛的種子是藉由螞蟻來進行傳播，根據長期野外觀察及定點攝影，九種螞蟻被認定具有傳播大花細辛種子的能力，相較於脊椎動物的傳播距離，螞蟻傳播的距離顯得較短，在本研究中的平均傳播距離為 77.5 公分。本研究也證實了種子上的油質體在螞蟻傳播上扮演極為重要的角色。此外，藉由分布位置的分析，顯示螞蟻蟻巢的分布與細辛幼苗的分布具有高度的相關性。因此，大花細辛的分布模式深受螞蟻傳播模式的影響。綜合上述的研究結果，大花細辛在繁殖的策略上顯現了兩項重要的機制，一為兩種共存的授粉系統；二為以螞蟻為媒介的傳播機制，此兩項關鍵性機制的作用之下，造就了大花細辛成功的繁殖。. V.

(9) Abstract Pollination and dispersal are two critical steps among plant reproduction. Delicate and diverse mechanisms on these two steps are derived from the results of coevolution. But these mechanisms are still unclear and full of uncertainty due to the lack of researches. Therefore, detailed and extensive studies on pollination system and dispersal pattern are helpful to document the population dynamic, delicate reproductive strategies and mechanisms. Plants in genus Asarum bloom and set fruits near the ground. Its pollination biology is full of speculations due to the extraordinary habit. Besides, distribution of small and isolated population may be hugely influenced by seed-dispersal pattern. In brief, we take Asarum macranthum as our material in this study to investigate on these two reproductive steps: „pollination‟ and „seed dispersal‟. Plants used to attract their pollinators by mimicry. The strategies are involved not only with food and sexual deception, but also with brood site imitation. Vogel raised a hypothesis of Asarum pollination. The ridges inside the calyx tube of Asarum have similar morphology with gills of basidiomycetes. They deceive fungus gnats to translocate the brood site and to help pollen transfer. No study with further statistical data can prove this hypothesis. Therefore, this study attempts to realize the pollination biology of Asarum macranthum and to prove Vogel‟s hypothesis. Moreover, we use the statistical analysis to testify the relationship between oviposition state and fruit ratio. One study area is set in the low altitude mountain of Northern Taiwan for long-term observation. By means of reproductive system, floral morphology and receptivity test, I conclude that Asarum macranthum is a protogynous and self-compatible species without any stamen movement. It most relies on pollinators to achieve pollination. Fungus gnats are discovered to oviposit on the ridges inside the. VI.

(10) calyx tube, fully supporting Vogel‟s hypothesis. Fungus gnat is proved to be the important agent of Asarum macranthum. This study also proved the oviposition state is positively correlated with the fruiting phenomenon by Fisher‟s exact test. Volatile analyses also prove that similar chemical substances are emitted between A. macranthum and fungus, which is another evidence to support the pollination hypothesis of brood site imitation. Besides, this study also observed the isopods-pollination phenomenon for the first time. Dark and dampness environment inside the calyx tube provide a shelter for isopods. The intrafloral behavior of isopods is helpful for pollination. The combination of two pollination systems enhances the fruiting ratio of A. macranthum in the field. A. macranthum is proved to be a myrmecochorous plant. By field observation and videotaping, nine ant species are recognized as the seed dispersers. Average dispersal distance is 77.5 cm, which is relatively shorter than the distance that dispersed by vertebrate animals. Seed removal rate decreased significantly when elaiosome is removed. Distribution analysis indicated that the distribution of Asarum seedlings is strongly correlated with the distribution of ant nests. Population dynamic and distribution of A. macranthum are strongly influenced by myrmecochorous phenomenon. Combining results from the above, A. macranthum presents dual-pollination system and myrmecochory on the reproductive strategy. Two critical mechanisms contribute to the successful reproduction.. VII.

(11) General Introduction Plant‟s sexual reproduction is a way to pass their genetic materials to the next generation, and it also plays a requisite role during life cycle. Successful reproduction can maintain the population‟s propagation and lead to flourishing. Among every step of reproduction, “pollination” and “seed dispersal” are two decisive and critical steps on reproductive process. These two steps are frequently concerned with biotic vectors, which are highly fluctuated in their population size in natural environment. Any mistake could influence the outcomes of reproduction, in terms of the fate of the individuals, and even the destiny of whole population. Therefore, detailed investigations on pollination biology and dispersal mechanism are extremely needed to document the reproductive strategy and population dynamics of plants. However, plants in genus Asarum bloom near the ground. Dark purple to reddish flowers are lack of nectars. Cryptic color and nonreward floral features make its pollination strategy interested to scientists. In addition, the spatial pattern of Asarum is clumped, formed a small and restricted population. Dispersal mechanism and agents may act as a major reason leads to such clumped spatial pattern. Therefore, this study aims to investigate the pollination and seed dispersal of plants in genus Asarum, and we use an endemic plant in Taiwan, A. macranthum, as our material to document its reproductive biology.. 1.

(12)  Part 1  Pollination Biology of Asarum macranthum. 2.

(13) Introduction Suits of floral characteristics that have coevolved with different types of pollinators are recognized as “pollination syndromes” (Ollerton et al., 2009). For example, flower characterized by white color, long spur, anthesizing at night and strong fragrance are linked to the phalenophily (moth-pollinated). Tubular flowers with abundant nectar and red or vivid color are intuitively associated with ornithophily (bird-pollinated) (Van der Pijl & Dodson, 1966). However, sapromyiophily (fly-pollinated) is most extraordinary and mysterious. The flowers with sapromyiophily syndrome usually imitate decaying substances, dung, or carrion to deceive flies coming for food or oviposition. The visiting behavior enhances the probability to achieve pollination (Van der Pijl & Dodson, 1966). Some angiosperm families, such as Asclepiadaceae, Aristolochiaceae, Rafflesiaceae, Hydnoraceae, Araceae and so on, have been recognized as sapromyiophilous (Van der Pijl, 1960). Among these families, members in family Aristolochiaceae show fabulous and varied morphological adaptations. The genus Aristolochia shows a brood-site imitation. With stinky odor, dull floral color, filiform appendage and dampness enviroment inside the calyx tube, plants in genus Aristolochia mimic the breeding site of its pollinators (flies or beetles). Furthermore, in order to increase the staying time, calyx tube with downward pointing hairs or wrong sign of illumination are presented. Therefore, pollinators are attracted to the traps and lay eggs within flowers to achieve pollination (Sakai, 2002). However, genus Asarum, within the same family, is distinctly different in habitat and floral morphology from Aristolochia. The flowers of Asarum bloom near the ground. Three dark-purple to reddish sepals unite as a calyx tube and there is no trap structure to detain its pollinator. Therefore, genus Asarum may exhibit the different pollination strategies.. 3.

(14) Several scientists tried to figure out the pollination strategy of Asarum. Peattie (1940) in his paper “How is Asarum pollinated?” summerized several scientists‟ view of the Asarum pollination and his own field observations. Some authors speculated that the pollinator of Asarum was slug or ant. Some authors strongly believed that the nocturnal crawling insects were the main pollinators. Some even took Myzus as pollinator. But not a single insect had been observed in Peattie‟s own observation. Therefore, the lack of long-term observation and empirical data contributed to the incongruent views of Asarum pollination. Taxonomically, Asarum is divided into two sections based on their morphology (Kelly, 1997). Section Heterotropa, has connate sepals (synsepaly), free styles, and short stamen filaments. While section Asarum, has free sepals (chorisepaly), connate styles, and long stamen filaments. These two sections are different in floral morphology which may exhibit to different pollination strategies. However, limited studies are available, only seven studies on section Asarum (Kugler, 1934; Wildman, 1950; Werth, 1951; Daumann, 1972; Vogel, 1978; Lu, 1982; Mesler and Lu, 1993), and only one on section Heterotropa (Sugawara, 1988). Therefore, the detailed pollination mechanisms of these two sections are still remaining unclear. But this study can get a rough idea about their different pollination strategies. At first, section Asarum, with a long stamen filaments, is dominated with a delayed autonomous self-pollination strategy- stamen filaments are bent to 90 degree that results a direct pollen deposition on the stigma (Lu, 1982; Kelly, 1997). But in section Heterotropa, with short filament and spatial isolation between stamen and stigma, pollens are necessarily transferred through pollinators (herkogamy) (Kelly, 1997). Vogel (1978) proposed a hypothesis of Asarum pollination. The flowers of Asarum imitate fruiting bodies of basidomycetes. Fungus gnats, whose larvae feeding on fungi, are attracted to translocate their mating and oviposition sites to Asarum. 4.

(15) flower. Therefore, the mating and egg-laying behavior of fungus gnats become a manner assisting pollen transmission. Several studies also discover same egg-laying behavior inside the calyx tube. However, most studies are on section Asarum (Mesler & Lu, 1993), only one research is involved with section Heterotropa (Sugawara, 1988). But none of them reveal the actual correlation between oviposition state and successful pollination by statistical data. How do plants attract flies to lay eggs? Dafni (1984) suggests the major attractants that flowers mimic the brood-site to their pollinators is olfactory. This phenomenon may be termed as “chemical mimicry”. Therefore, if Vogel‟s hypothesis is true, does Asarum show chemical mimicry to their pollinator- fungus gnats- as well? Therefore, scent analysis seems to be a suitable tool to answer this question. Several studies have already applied scent analysis to survey the chemical compound compositions of plant odor and use them for further investigations (Schiestl, 2000; Ayasse, 2000). In this study, I attend to study the phenomenon of sapromyiophily and deceptive pollination strategy on genus Asarum (especially section Heterotropa) to realized its mysterious pollination system. Therefore, A. macranthum was applied as our material to illustrate its breeding system and check the Vogel‟s hypothesis. Furthermore, I also try to show the relationship between egg-laying behavior and successful pollination. In brief, this study has two principle aims: (1) Document the floral biology, breeding system and pollination mechanism of Asarum macranthum. (2) Prove Vogel‟s hypothesis by surveying the plant-pollinator relationship of brood-site imitation.. 5.

(16) Material and Methods Species and study site Asarum macranthum (fig 1), which is endemic to Taiwan, is distributed on forest floors and margins at the altitude of 400 to 2000 m (Huang, 1996). Individual plant of A. macranthum usually occurs as a clump, and each plant bears one to several flowers at ground level. Three dark-purple to reddish sepals united as a calyx tube. Each calyx lobe has several white lamellae on base. Our study site was located at “Mt. Ergo” on the border between Wenshan District (Taipei City) and Shiding Township (Taipei County). A 3 x 13 m2 study area was set at Mt. Ergo for a long-term field pollinated observation and experiment. Additional fresh materials collection and phenology observation were processed at “Mt. Tatung” (Wulai Township, Taipei county). Several samples collected from both sites were transplanted to greenhouse of Department of Life Science, National Taiwan Normal University, Taipei, Taiwan for scent analysis Breeding system experiment Plants from field and greenhouse were assigned for tests of nature fruit set, autonomous self-pollination and self-compatibility to conclude the breeding system of A. macranthum. (1) Natural fruiting rate (in field): In 2009 & 2010, every calyx tubes of A. macranthum were marked in the 3 x 13 m2 study site when they were still in bud. After two months, the state of ovary (swollen or abort) was checked to count for the number of successful fruiting and to estimate the fruiting rate. (2) Bagged fruiting rate (in greenhouse): In 2008, 2009 & 2010, 10 flowers (1 flower per plant) which were still in buds were randomly selected, and then bagged with mesh net to isolate pollinators‟ interferences. Two weeks after blossom, when flowers already lost all their pollinated receptivity (personal observation), mesh. 6.

(17) nets were removed to avoid affecting the fruit development. After two months, the state of ovary (swollen or abort) was checked to count for the number of developed fruits and to estimate the fruiting rate.. Figure 1– Habitat and morphology of Asarum macranthum A- Habitat. B- Flower, three sepals united into a calyx tube, white lamellae between orifice and three calyx lobes. C- Cordate leaf, always with white spotted and sparsely hair. D- Flower buds. E- Longitudinal section of Asarum macranthum. White triangle symbol indicates the pistil. Yellow arrows indicate the stamens. Star symbol indicates the colorless translucent area- “window pane”.. 7.

(18) (3) Hand-pollination for testing autogamy: In 2008 & 2010, in order to reveal the self-compatibility, fresh and high viability pollens* were smeared on the stigma of the same flower. Finally, bagged all the treatment flowers with mesh net. After two months, state of ovary was checked to count for the number of developed fruits and to estimate the fruiting rate. *. Pollens were used just after anthers dehisced through the second day, pollen grains were well-stained with acetocarmine test (any pollen that stained in red reflects its high viability).. (4) Hand-pollination for testing allogamy: In 2008 & 2010, fresh and high viability pollens were smeared on the stigma. ＊. of the different flowers. After that, all the. treatment flowers were bagged with mesh net. After two months, the state of ovary was checked to count for the number of developed fruits and to estimate the fruiting rate. ＊. Stigma of different flowers were kept for two days age, still remaining highly. receptivity according to aniline blue staining (any pollen tube that germinates into pistil can by stained in blue by aniline blue) (Appendix).. Reproductive table construction In order to document the whole reproductive process thoroughly, a digital camera was set to record morphological change of calyx tube and the state of two whorls of anther dehiscence during anthesis. Furthermore, two kinds of staining methods were applied to evaluate the viability of pollen and stigma in different stages. (1) Morphology observation: One flower that still in bud was selected for continuous observation. Digital camera was used to take pictures every morning for 7 days to record the morphological changes of calyx tube. Another bud was also chosen for stamen observation. Parts of the calyx tube (about 1.5 x 1 cm2) was cut off to see. 8.

(19) the dehiscence of stamens clearly and use digital camera to take pictures every morning for 7 days to record the morphology changes of stamens. (2) Pollen viability: Two drops of 0.5% acetocamine was added on the slide, and immersed pollens in the droplet. Waiting for two minutes to completely absorb, a cover glass was put on it, and observed the slide with microscope to check for the stainability. Any pollen that stained in red reflects its high viability. Pollens with different maturation stage (dehiscence form anther for 1~6 day) were tested according to the procedure above. The degree of stainability can reflect the viability of pollens. (3) Pistil receptivity: In vivo pollen germination was used to evaluate the receptivity of pistil. Fresh and viable pollens (all dehisce from anther for two days) were smeared on stigma of different maturation stage (1~6 days since calyx tube opening). Waiting for 4 hours for pollen tube germination, aniline blue staining methods (Appendix) was applied to stain the pollen tube that grow into the pistil. I put stained stigma on the slide and added two drops of DABS solution, and then put on a cover glass and observe the slide under an UV microscope. Stained pollen tubes excited by UV emitted a green light. By surveying the condition of germination, the pistil receptivity of different maturation stage can be evaluated.. Pollinator observation Four-year field observations were processed between 2007~2010, Two kinds of treatments were used to ensure the pollinator compositions and visiting frequency. First, in 2007 &2008, several calyx tubes were cut at pedicels, put them in the plastic bag immediately and took them back to the laboratory to examine any visitor in the calyx tubes. By the same time, recorded their visiting frequency and pollen load on their bodies. Second, in 2009 & 2010, a digital video recorder was set to record the. 9.

(20) visiting behavior during anthesis. During these two years, from February to March, 24 hours (4 hours a day) were recorded to quantify the visiting frequency of these visitors. All visitors were killed by diethyl ether, and preserved under dry condition for six month. After that, the samples were taken out for ion-coating for four minutes, and observed for any pollen load on the bodies under scanning electronic microscope.. Pollinator behavior With the preliminary observations on pollinators from 2007 to 2010, fungus gnat (family Sciaridae) and isopod are recognized as pollinators. In order to reveal their behavior inside the calyx tube, several calyx tubes were taken for observing the presences of eggs and remnants. Moreover, the correlation between oviposition and fruiting rate was evaluated in this study. Several calyx tubes were taken an annular cut (without harm to the ovule) and brought back to the laboratory (115 in 2009, 156 in 2010) to examine if any eggs exist in the inner ridges. Flowers were labeled in the field, and checked the state of ovary after one month. In the end, fisher‟s exact test was applied to analyze the correlation between fruiting rate and egg laying state to make sure their relevance. Among these dissected calyx tubes which have eggs inside, 28 calyx tubes were randomly chosen for further analysis. Every cross-section of calyx tube was photographed with digital camera through stereomicroscope. In order to reveal that oviposit place was corresponding to the place of stigmas and stamens, the locations of eggs were recorded by three main parts separately (fig 2). These three areas were distinguished by two lines (one is the upper edges of the white patches; the other is three blocks away from the first line). The calyx was separated into “Upper area”, “Pistil area” and “Anther area” in order (fig 2). These three areas correspond to the. 10.

(21) region of upper calyx, stigma, and stamens respectively. All the data were analyzed with chi-square test to know the preference of egg-laid regions. Furthermore, revealed the relationship between ovipositing region and reproductive organs.. Figure 2– Three separate areas of dissected calyx tube. Scent glands observation In order to realize which region of A. macranthum emits the scent, scanning electronic microscope was used to visualize the gland cells. The gland cells response for scent emission are called “osmophores”. Whole calyx tube can be divided into 5 parts, namely calyx lamella (CL), dorsal orifice (DO), ventral orifice (VO), upper calyx (UC), and lower calyx (LC) to evaluate the distribution and density of osmophores (fig 3). Each of them was fixed in 70% ethanol, and then transferred sequentially to 85%, 95%,. 99.5%. ethanol,. and. 100%. acetone. for. dehydration. All the samples were critical-point dried (CPD), then put on the aluminum stub, and finally, ion-coated with gold-palladium for observation under scanning electronic microscope (SEM). Detailed structure of osmophore was examined in 25x and 100x.. Figure 3– Osmophore evaluation zone Calyx tube is cut into 5 parts to evaluate the osmophore location. These parts were named by their abbreviation (CL-calyx lamella, DO-dorsal orifice, VO-ventral orifice, UC-upper calyx, LC-lower calyx). 11.

(22) Volatile collection Plants used for volatile collection were selected from greenhouse that collected from two study site. Five bloomy flowers were placed in a closed acrylic chamber. Air was extracted from the chamber by a battery-operated vacuum pump (SKC). Two different absorb tubes were used for trapping the volatile, one is activated charcoal tube and the other is Tenax TA tube. Flow rate was control at 540ml/min in charcoal tube, 385ml/min in Tenax TA tube. Before use, the Tenax TA was cleaned with 200 μl dichloromethane. Ambient air was also collected as a control sample to avoid background contamination. After volatile collection, samples were sealed and stored at 4℃.. GC-mass spectrometry The volatiles trapped on the adsorbent were eluted with 300 ml diethyl ether, and 2 ml of eluent was used for GC–MS analysis. The analysis was performed using a GC-17A gas chromatograph coupled with a GC–MS-QP5000 ver. 3 mass detector (Shimadzu, Kyoto, Japan). A DB-1 fused-silica capillary column (30 m; inner diameter, 0.25 mm; film thickness, 0.25 mm) was used. The injection temperature was maintained at 40℃ for the first 5 min, programmed to increase by 5℃/min to 250℃, and held at 250℃ for 5 min. Helium was used as the carrier gas. After each analysis a 1-ml aliquot of nonyl acetate solution (0.5 mg/ml dichloromethane) was added to the same sample as an internal standard to calculate the amounts of the volatiles and then immediately analyzed under the same conditions. The volatile compounds were identified by comparing their GC retention times and MS spectra with those of the authentic compounds, or tentatively identified by MS spectra in the NIST 02 mass spectral library.. 12.

(23) Results Breeding system In both autogamous and allogamous artificial manipulations, no significant difference (chi-square test, 0.75<p<0.5) between the fruit sets indicated that A. macranthum is highly self-compatibility (Table 1). Fruit set is about 50% in field and 0% in bagged flowers means that pollen grains are necessarily transported by pollen vectors. Autonomous self pollination which has been found in species of subgenus Asarum, e.g. A. caudigerum, is absent in this species. Table 1–Pollination manipulations of Asarum macranthum. Reproductive table (1) Morphology change: From day 0 to day 7, three calyx lobes started to dehisce from each other, extended to a plane, and curved backward until touched to the calyx tube (fig 4-A). 12 stamens arranged in two whorls (fig-5) which matured at different time. Inner whorl of anthers dehisced at day 1, and outer whorl of anthers have to wait until day 3 to release pollens (fig4-B). Two whorls of anthers are lack of movement during anthesis. (2) Pistil receptivity: A. macranthum is a protogynous plants, pistils are highly receptive at the beginning when anthesis. Constant receptivity maintained from day 0 to day 7 (fig4-C) that pollen tubes can germinate into stigma tissues (fig 6). (3) Stamen viability: Pollen grains of both whorls of anthers were all stain in red for a. 13.

(24) week, which indicated high viability from day1 to day 7 (fig4-D). Moreover, outer whorl of anthers dehisced latter, that make longer duration of releasing viable pollens.. 14.

(25) *. Figure 4- Reproductive table of Asarum macranthum A- Morphology change of calyx tube. Three calyx lobes extend, and then curve backward until touch to the calyx tube. B- Two whorls of anthers dehisced at different time. The inner whorl of anthers dehisced at day1. However, the outer whorl of anthers dehisced until day 3. C- Pistils are highly receptable since anthesis, and the receptivity maintain for about 1 week. D- Pollens are viable from day 1 to day 7. Pollens of both whorls of anthers reveal no difference on their viability. *-. Because morphology and receptivity hold constant from day4 to day 7, we just combine the data together (day4~day7) and not show it separately.. 15.

(26) Figure 5– Arrangement for two whorls of anthers Twelve anthers of Asarum macranthum arrange in two whorls. Star symbols indicate the pistil. White arrows indicate the outer anther. Yellow dash arrows indicate the inner anther.. Figure 6– Pistil receptivity test – aniline blue staining From day 0 to day 7- stigma maintains high receptivity. Pollen tubes can germinate into stigma.. 16.

(27) Pollinator observation Preliminary visiting data (table 2 & 3) indicated that many terrestrial invertebrates and fungus gnat are main visitors. These visitors were recognized as legitimate pollinators by checking pollen load and visiting frequency. The results show that “isopod” and “fungus gnat” are recognized as the pollinators of A. macranthum (Fig7-A &B, respectively). These two agents were with higher visiting frequency and pollen loading on the body (fig 8 & fig 9) than any other visitors (including Arachnid, Millipedes and Collembola). In 2009 and 2010, from the images that captured from digital video recorder, isopods hide into the calyx tube as a shelter (fig 10) and being active in the night around the calyx tube (fig 11). On the other hand, fungus gnats (family Sciaridae) were also found to copulate in the air (fig 12-A). Female fungus gnat entered the calyx tube to lay eggs (fig 12-B~F) (male fungus gnat sometimes entered the calyx tube too). These intrafloral movements are the critical steps for pollination. Besides, some fortuitous incidents happened. For example, an ant nest was built in calyx tube once. Over ten workers and numerous larvae are found covering with pollens. Spiders (Aranchnid) can also be observed hide in calyx tube to wait for their prey. These behaviors may achieve pollination, but they are excluded as pollinator for their inability for carrying pollens. Combing the behavior inside flowers, visiting frequency and pollen load, “isopods” and “fungus gnat” are recognized as the legitimate pollinators of A. macranthum. In other words, A. macranthum exhibits a dual pollination system.. Figure 7- Legitimate pollinators of Asarum macranthum A- “Isopods”, abundant in the organic soil. Usually craw along the litters, and always found within the calyx tube. B-. “Fungus gnat”, a dipteral insect, always come into the calyx tube for oviposition.. 17.

(28) Table 2- Number and pollen load on visitors caught in the calyx tube. Table 3- Visiting frequency and time (recorded by digital video recorder). 18.

(29) Figure 8- Isopod- images of scanning electronic microscope A~C- SEM image of isopod in 18x, 100x, 900x. Pollens adhered on the pereon. D~F- SEM image of isopod in 18x, 50x, 900x. Pollens adhered on the pereon.. Figure 9-Fungus gnat- images of scanning electronic microscope A~C- SEM image of fungus gnat in 50x, 150x, 900x. Pollen adhered on the abdomen. D- SEM image in 18x. E&G- SEM image in 150x, 900x. Pollen adhered on the halter. G&H- SEM image in 150x, 900x. Pollen adhered on the leg.. 19.

(30) Figure 10- Video images of the pollinator “Isopods” A~C – Three continuously frames show that the isopods craw into the calyx tube D – Another frame of the isopods craw into the calyx tube. Figure 11- Video images in night vision of the pollinator “isopods” A~D- Isopods are the nocturnal animal, they craw along the litters, and sometimes get into the calyx tube. Frequently visitations are observed during the long-term videotaping.. 20.

(31) Figure 12- Video images of the pollinator “fungus gnat” A- Male and female fungus gnats first copulate in the air. B&C- Male fungus gnat gently push the female fungus gnat into the calyx tube.. 21.

(32) Figure 12- Video images of the pollinator “fungus gnat” D~F- Intrafloral movement of fungus gnat. F- Fungus gnat slowly craws out the calyx tube. 22.

(33) Pollinator behavior In our observations, fungus gnats copulated in the air and entered the flowers. After examining for oviposition (115 flowers in 2009, 156 in 2010), there are about 25% calyx tubes had eggs inside (21.2% in 2009, 26.3% in 2010). Eggs were laid in the grids surrounded by ridges, and generally 1~5 eggs are laid in a calyx tube (Fig 13). Fisher‟s exact test was shown that the flowers with eggs inside have significantly higher successful fruiting rate (p=0.0016 in 2009, p<0.0001 in 2010) (fig 14). It means that egg-laying behaviors contribute to successful fruiting. And successful fruiting rate are directly associated with the successful pollination. Different egg-laying areas were recorded inside 28 calyx tubes. 4 calyx tubes with eggs laid in upper area (14.3%). 24 calyx tubes with eggs laid in pistil area (78.6%). 4 calyx tubes with eggs laid in anther area (14.3%) (figure 15). Sum of percentage are larger than 100%, because some calyx tube with more than two locations were being laid. Significant differences (chi-square, p<0.0001) are among three egg-laying areas (fig 15). On the other hand, isopods were found to hide inside the calyx tube, which are functioned as a shelter for isopods to hide and shed their exoskeleton (Fig 16). As they hide inside the calyx tube, narrow space will force them to touch the stigma, and the pollen grains are transferred from their body to the stigma as well.. 23.

(34) Figure 13- Eggs of fungus gnat laid in the calyx tube. Figure 14- Fisher‟s exact test – ovipositing and fruit set correlation In the chart, y-axis is the fruiting ratio, and x-axis is the egg laying or not inside the calyx tube. There is a significantly relationship between egg laying and fruit set (p=0.0016 in 2009, p<0.0001in 2010). Figure 15- Chi-square test of egg-laying area Significant differences are among three egg-laying areas (p<0.0001). Charts marked by the same letter are not significantly different.. 24.

(35) Figure 16- Behavior of isopods inside calyx tube A- Isopods usually hide in the calyx tube. B- Red circle is the place that isopods hide. C- Isopod is moulting inside the calyx tube. D- Pollen grains are adhering at the cephalothorax and pereon.. Scent glands observation Surveying all the samples from 5 different parts of calyx tube under scanning electronic microscope, numerous scent glands (osmophore) were observed to present in the lamellae and ridges inside the calyx. In the “CL” region, every lamella is covering with osmophores, but none is on the three sepals. In the “DO” and “VO” regions, osmophores are largely gathered in the orifice ring. Inside the calyx tube, “UC” and “LC” regions are dominated by their net structure, every net ridges and the top of small process are covering with osmophores, especially in the bottom of “LC” part, which is overwhelming by osmophores (fig 17). By surveying the distributions of osmophore, these scent glands were spread out inside the calyx tube, which may function as an attractant to pollinators. With the highest density of osmophores on the base inside the calyx tube, strongest odor may be the reason why pollinators go downward and get the pollen attached to their bodies.. 25.

(36) Figure 17- The scent glands location of the Asarum macranthum A~E- Each frame contains two graphs inside. They are the same region with different magnifying power. Right-25x, Left-100x. A-Region of calyx lamella (CL).. B-Region of dorsal orifice (DO).. C-Region of ventral orifice (VO). D-Region of upper calyx (UC). E-Region of lower calyx (LC). F-Individual osmophore magnify in 1700x.. 26.

(37) Scent analysis The scent chromatogram of A. macranthum is presented in fig 18. Both Asarum scent and blank air show similar pattern in the mass spectrum. However, about 3 peaks, 7.861min, 8.130min and 8.404 min respectively are obviously different in their abundance of volatile. In 7.861min, volatile and blank air chromatograms were compared to avoid background interference (Fig 19). The major background interferences were appeared in molecular weight 64, 76, 77, 78. After eliminating these interferences, a new volatile chromatogram is presented (Fig 20). Volatile compounds were identified by comparing the GC retention time and MS spectra. Finally, similar structure was compared with a monounsaturated fatty acid “oleic acid” (Christie, 2005) (Fig 21). Both of them have a similar relative abundance on the molecular weight 55, 69, 83, 97, 111, 125, but different in their maximum molecular weight (204 in Asarum scent, 264 in oleic acid). These results reveal that the molecular structure of the odor emitted by Asarum is similar with the oleic acid, but with a short-chain. In 8.130 min and 8.404min, nothing can be identified resembling structure by comparing the online volatile database and electronic library. (Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland.) (http://lipidlibrary.co.uk) Further studies and experiment designs are needed to solve these problems in the future.. Figure 18- Scent chromatogram of Asarum macranthum and blank air Similar pattern are shown in the scent chromatograms between Asarum macranthum (A) and blank air (B). Three peaks are obviously different. They are found in different time. a-. Scent in 7.861min. b- Scent in 8.130min c-. Scent in 8.404min. 27.

(38) Figure 19- Scent chromatograms in 7.861min Scent chromatogram comparison between Asarum macramthum (A) and blank air (B) shows different patterns on their mass spectrum. Any peak in common is regard as background interference. And we found the major background interferences appear in molecular weight 64, 76, 77, 78.. 28.

(39) Figure 20- New volatile chromatogram after eliminating background interferences After eliminating the background interferences on peak 57, 64, 76, 77, 78, we get a new volatile chromatogram. Obvious peaks are shown in molecular weight 55, 69, 83, 97, 105, 111, 119, 125. Similar peak relative pattern are shown in molecular weight 55, 69, 83, 97, 111, 125 (marked by arrow) with oleic acid (fig 21). The maximum molecular weight is on the peak 204.. Figure 21- Volatile chromatogram of oleic acid (modified from the figure in Christie, 2005) Oleic acid is dominated on the peaks 55, 69, 83, 97, 111, 125 (marked by arrow). The maximum molecular weight is on the peak 264. This chromatogram is modified from the electronic library. 29.

(40) Discussion Breeding system predominance A. macranthum, with stable and about 50% fruit set in field, is attributed to several delicate designs on reproductive system. First of all, according to the reproductive table (fig 4), two whorls of stamens are matured at different time, caused prolong male phase to provide viable pollens. Second, A. macranthum is a protogynous plant which adapted for cross-pollination. However, receptivity and viability of stamens and pistils remain an overlap periods that imply a potential of self-pollination. In other words, A. macranthum has the possibility of both self and cross pollination. These breeding system predominances are well-cooperated in A. macranthum.. Dual-pollination system Numerous studies are trying to find out the effective pollinators of Asarum. But without long-term field observation and suitable experiment design, these speculations might just be the misinterpretation on incident. Vogel (1978) discovered that some fungus gnats (Mycetophila) come into the flowers as they open, and intrafloral movements enhanced the successful pollination. This interesting and critical observation opened the prologue of Asarum pollination. Numerous studies were involved in this topic. Some of them failed to discover such a sapromyiophilous pollination system (Lu, 1982; Tanaka & Yahara, 1987), and some of them documented the egg-laying phenomenon (Sugawara, 1988; Mesler & Lu, 1993). Consequently, in this study, based on the efforts of previously study, this study has been successfully realized the pollinator composition and their behavior contributed to the pollination through a long-term field observation. This study has shown the pollinators of A. macranthum are “fungus gnats” and “isopods”. These two pollinators show constant and regular visitation when A. macranthum blooms. Detailed relationships between. 30.

(41) Asarum and pollinators are going to discuss below.. Pollination system 1 – “deception” A. macranthum exhibits a specific relationship with their pollinators. Unlike its related genus Aristolochia, A. macranthum is lack of trap to detain pollinators. It should have an alternative strategy to attract pollinators. In this study, Vogel‟s hypothesis is proved by long-term observations and experiments. Based on our observations, fungus gnats copulated outside and female fungus gnat came into the calyx tube to lay eggs. With similar shape inside calyx and damp environment, A. macranthum successfully attracts fungus gnat with brood-site imitation. Furthermore, in this study, two experiment designs have shown the relationship between egg-laying behavior and successful pollination. First, fisher‟s exact test shows that there is a significant relationship between egg-laying and fruiting rate. In other words, this ovipositing behavior assisted to successful pollination. Second, significant relationship was shown that much more eggs were laid in pistil zone. This phenomenon may explain flies spent more time in the pistil zone that will enhance the opportunity of pollination. With these two evidences, I believe that the behavior of fungus gnat could be critically important among the pollination relationship. Eggs inside the calyx tube may hatch, but they do not feed on the tissues of calyx, causing the larvae never getting beyond the first instar (Proctor, 1996). Some study even said that the tissue of Asarum is poison to gnat larvae (Vogel, 1973). If fungus gnats didn‟t benefit from Asarum flower, this relationship should be eliminated through natural selection. But why this deceptive strategy could maintain stable and sustained? Some researches provide an issue that pollinators are passive in this relationship, all the adaptations are just on the part of the plant. In this case, three explanations could apply on this phenomenon. First of all, Asarum blooms in early spring. It is the time which fungi are scarce. Fungus gnats may easily been deceit and come into the calyx tube. Second, the life cycle of fungus gnat are very short, about 4. 31.

(42) weeks are spent from egg to adult. Therefore, much of the adult fungus gnats may be inexperienced, they could not look for a suitable brood site through learning. Third, Females flies lay less than 20 eggs at a time, and up to 100~300 eggs in total, but there are just averagely about 2~5 eggs found in one calyx tube. Small portion of deceit eggs should not cause a tremendous effect on flies‟ population propagation. Therefore, three explanations and wonderful imitation of A. macranthum contribute to a stable deceptive pollination system.. Pollination system 2 –“shelter” Shelter-pollination strategies are usually used by some food-rewardless plants. These plants provide a resting place for pollinators. For example, petals and sepals of Serapias vomeracea form a small tube. The insects use this „floral tube‟ to rest on or as a shelter, and then help pollen transfer (Pellegrino et al, 2005). Similar shelter-pollination strategy is also shown in this study. Except fungus gnat, isopod is another agent recognized as legitimate pollinator of A. macranthum in this study. To our knowledge, this agent is never reported by any other researches, and it should be a new kind of pollination phenomenon. Based on our observations, isopods are found hiding inside the calyx tube frequently, and even moulting as well. Through the images that captured by the digital video recorder, most isopods are active mainly in the night. They crawled along the litters and sometimes will get into the calyx tube. In the morning, isopods hide in the litters and some of them will stay in the calyx tube, utilizing it as a shelter. When isopods passed in and out, pollen grains adhere to their cephalothorax and pereon (fig 8). With frequent visitation and appropriate body size, isopods successfully transferred pollens around the Asarum flowers. Therefore, isopods are recognized as the pollinator of A. macranthum. This is the first time that isopods are recognized as legitimate pollinator.. 32.

(43) Egg-laid preference An interesting phenomenon was discovered when I dissecting the calyx tube of A. macranthum to evaluate the ovipositing behavior. Most of eggs are found to be laid in pistil area. Is there anything attractant or mechanical structure in pistil area cause this difference? A similar phenomenon from some Aristolochia spp. was reported in Panama (Sakai, 2002). Eggs are laid most around the gynostemium inside the calyx tube. “Window pane” seems the structure to cause such differences. Window pane is a colorless translucent area at the bottom of the trap (fig1-E), surrounded by a darker pigmentation. According to Hilje (1984) and Cammerloher (1923), flies are photoactically oriented by light color. Therefore, pollinators fly toward the direction, and get trap around the gynostemia. That is why most of eggs are laid around gynostemia. Similar structures are present in A. macranthum, window panes are exist at the base of the calyx tube and just beneath the pistil zone. Another attractant may be the osmophores, according to fig 17, numerous osmophores present on the ridges inside calyx tube. Especially in the bottom of the “LC” part, which are overwhelmed by osmophores, and that is exactly the place of window pane. With the strongest odor, flies are attracted downward to lay eggs. Moreover, according to Proctor (1996), pale patches in the Asarum flower are much more dampness than the neighboring regions. Because of pale patches contribute to a much higher rate of transpiration. Therefore, fungus gnats are attracted to this dampness site (white patches) to lay eggs. Finally, a morphological structure may be another reason to cause such difference. There is a shrink between pistil zone and anther zone, only some small space can pass through. Therefore, pollinators are usually detained in the pistil zone. In short, there are four reasons that cause pollinators for staying, even laying egg in the particular pistil zone. First is the window pane to deceit pollinators to fly toward. Second, numerous osmophores covered at the base of the calyx tube and act as a strong attractant. Third, the dampness sites imitated the gills of fungi. And forth, a shrink in the calyx tube cause pollinators to detain. Four kinds of imitations involve with odor, illumination,. 33.

(44) morphology, and dampness contributed to the specific preference on the choice of egg-laying region.. Scent imitation No studies have described obvious floral scent emission in Asarum species until Azuma (2010) investigated the floral scent emission of seven Asarum species (including A. yaeyamense and its related species). Methyl tiglate is reported to contribute to the floral scent of A. yaeyamense. But in this study, instead of methyl tiglate, a chemical substance related to oleic acid is proved to be the major scent volatile of A. macranthum. Various combinations of floral volatiles presented in different Asarum species may reflect to different pollination strategies. In the case of A. macranthum, the pollination strategy should be the scent imitation with fungus. According to Vogel‟s hypothesis, Asarum flowers imitate mushrooms of Basidomycetes on the ridges morphology inside the calyx tube and also the damp environment (Vogel, 1978). However, scent imitation may be another critical feature in A. macranthum. In our experiment results, the odor composition of A. macranthum is the substance which is similar to the monounsaturated acid “oleic acid”. This substance, which is also one of the main fatty acids found in members of the Basidomycetes (Weete, 1974). Moreover, the fungi that mentioned in the Vogel‟s hypothesis are belonging to the family Boletaceae (Yinger, 1983). The fungi of this family are also dominated with oleic acid in their scent composition (Karine, 2006). This special chemical substance is involved in whether Asarum flowers or fungi, which may act as a crucial role in the brood-site imitation. Dafni (1984) said “The imitation of an oviposition substrate is mainly olfactory”. Therefore, the story goes to that the A. macranthum emitted the similar chemical odor of fungi, which functioned as a primary attractant to fungus gnat. And then, fungus gnats are attracted to enter the calyx tube. In the mean time, morphological imitation inside the calyx tube and damp environment are the secondary attractant to deceit fungus gnat. Finally, fake. 34.

(45) illumination and highest odor intensity around the gynostemium elongate the time that fungus gnat staying in the calyx tube. Multiple delicate and well-designed strategies successfully attract pollinators.. Successful pollination In sum, the pollination of A. macranthum is attributed to several delicate designs on four different phases. First, the breeding system predominance enhances the pollination possibility. Second, two kinds of pollinators exhibit good pollen-carrying ability and consistent visitation. Third, delicate morphological design, including window pane, osmophores, dampness environment and shrink of calyx tube strongly influence the behavior and choice of pollinators. Forth, similar scent volatile composition between Asarum and fungus shows a wonderful imitation and also strengthen the brood-site imitation relationship. All these strategies and adaptations are well-functioned and lead to successful pollination.. Pollination model for section Heterotropa In this study, A. macranthum is applied as a material to study its pollination strategy. All the adaptations, including ridges inside calyx tube, white patches as window pane, scent emission, shape of calyx tube, pistil and stamen morphology contribute to successful pollination and make this pollination system unique. In Taiwan, there are about 15 taxa in genus Asarum (about 10 taxa in section Heterotropa). Some of them differ in their shape of calyx tube, some lack of white patches inside calyx tube, some with long appendage on stigma and some with fewer ridges. Morphological variation between each taxa may act as a critical role on their pollination strategy. Therefore, in this study, the pollination biology of A. macranthum plays an important role as a model of pollination strategy for other different taxa among genus Asarum.. 35.

(46) References 1. Ayasse, M., F. P. Schiestl, H. F. Paulus, C. Löfstedt, B. Hansson, F. Ibarra, and W. Francke. 2000. Evolution of reproductive strategies in the sexually deceptive orchid Ophrys sphegodes: how does flower-specific variation of odor signals influence reproductive success? Evolution 54: 1995–2006. 2. Azuma, H., J. I. Nagasawa, and H. Setoguchi. 2010. Floral scent emissions from Asarum yaeyamense and related species. Biochem. Syst. Ecol. 38 (4): 548-553. 3. Cammerloher, H. 1923. Zur Biologia der Blüte von Aristolochia grandiflora Swartz. Ö sterr. Bot. Z. 72: 180-198. 4. Christie, W. W. 2005. The lipid library. http://www.lipidlibrary.co.uk 5. Dafni A. 1984. Mimicry and deception in pollination. Ann. Rev. Ecol. Syst. 15: 259-278. 6. Daumann, E. 1972. Die Braune Haselwurz (Asarum europaeum L.), ein obligator Selbstbestäuber. Preslia 44: 24-27. 7. Hilje, L. 1984. Fenologia y ecologia floral de Aristolochia grandiflora Swartz. (Aristolochiaceae) en Costa Rica. Brenesia 22: 1-44. 8. Huang, S. F. 1996. Asarum. In Huang T. C. et al. (eds.), Fl. Taiwan 2nd ed. Vol. 2. pp. 642- 651. 9. Karine, P., A. Paul, G. André, and J. T. Russell. 2006. Fatty acid composition of lipids from mushrooms belonging to the family Boletaceae. Mycol Res. 110: 1179-1183. 10. Kelly, L. M. 1997. A cladistic analysis of Asarum (Aristolochiaceae) and implications for the evolution of herkogamy. Amer. J. Bot. 84 (12): 1752- 1765. 11. Kugler, H. 1934. Zur Blutenokologie von Asarum europaeum. Berichte der Deutschen Botanischen Gesellschaft. 52: 348-354. 12. Lu, K. L. 1982. Pollination biology of Asarum caudatum (Aristolochiaceae) in northen California. Syst. Bot. 7(2): 150-157. 13. Mesler, M. and K. Lu. 1993. Pollination biology of Asarum hartwegii. 36.

(47) (Aristolochiaceae): an evaluation of Vogel‟s mushroom fly hypothesis. Madroño. 40: 117-125. 14. Mori, T., H. Kuroiwa, T. Higashiyama, and T. Kuroiwa. 2006. Generative Cell Specific 1 is essential for angiosperm fertilization. Natural Cell Biol. 8(1):64-71. 15. Ollerton, J., R. Alarco´n, N. M. Waser, M. V. Price, S. Watts, L. Cranmer, A. Hingston, C. I. Peter, and J. Rotenberry. 2009. A global test of the pollination syndrome hypothesis. Ann Bot. 103: 1471-1800. 16. Peattie, D. C. 1940. How is Asarum pollinated? Castanea. 5: 24-29. 17. Pellegrino, G., D. Gargano, M. E. Noce, and A. Musacchio. 2005. Reproductive biology and pollinator limitation in a deceptive orchid, Serapias vomeracea (Orchidaceae). Pl. Sp. Biol. 20:33-39 18. Proctor, M., P. Yeo, and A. Lack. 1996. The natural history of pollination. The New Naturalist Series. Timber Press, Portland, Oregon. 19. Sakai, S. 2002. Aristolochia spp. (Aristolochiaceae) pollinated by flies breeding on decomposing flowers in Panama. Amer. J. Bot. 89(3): 527-534. 20. Schiestl, F. P., M. Ayasse, H. F. Paulus, C. Löfstedt, B. Hansson, F. Ibarra, and W. Francke. 2000. Sex pheromone mimicry in the early spider orchid (Ophrys sphegodes): patterns of hydrocarbons as the key mechanism for pollination by sexual deception. J. Comp. Physiol. A 186, 567–574. 21. Sugawara, T. 1988. Floral Biology of Heterotropa tamaensis (Aristolochiaceae) In Japan. Pl. Sp. Biol. 3: 7-12. 22. Tanaka, H. and T. Yahara. 1987. Self-pollination of Asarum caulescens Maxim. (Aristolochiaceae) in japan. Pl. Sp. Biol. 2: 133-136. 23. Van der Pijl, L. 1960. Ecological aspects of flower evolution Ⅰ. Evolution 14: 403-416. 24. Van der Pijl, L. and C. H. Dodson 1966. Orchid Flowers: Their Pollination and Evolution. University of Miami Press, Miami. 25. Vogel, S. 1973. Fungus gnat flowers and fungus mimesis. In: Brantjes, N. B. M.. 37.

(48) and Linskens, H. F. (eds.), Pollination and Dispersal, 13-18. Nijmegan, Netherlands. 26. Vogel, S. 1978. Pilzmuckenblumen als Pilzmimeten. Flora. 167: 329–398. 27. Weete, J. D. 1974. Fungal Lipid Biochemistry: Distribution and Metabolism, Monographs in Lipid Research, Vol. 1. Plenum Press, New York. 28. Werth, E. 1951. Asarum europaeum, ein permanenter Selbstbefructer. Berichte der Deutschen Botanischen Gesellschaft. 64: 287-294. 29. Wildman, H. 1950. Pollination of Asarum canadense L. Science. 111: 551. 30. Yinger, B. R. 1983. A horticultural monograph of the genus Asarum senus lato, in Japan. Master thesis, University of Delaware. Newark, DE.. 38.

(49) Appendix-Aniline blue staining Mori, T., H. Kuroiwa, T. Higashiyama, and T. Kuroiwa. 2006. Generative Cell Specific 1 is essential for angiosperm fertilization. Natural Cell Biol. 8(1):64-71.. Recipe 1. Fixative: Acetic acid/EtOH(1:3)solution 2. EtOH series: 70%、50%、30%EtOH 3. Alkaline treatment solution(ATS): 8M NaOH 4. Decolorlized(*) aniline blue solution(DABS): 0.1%(w/v)aniline blue in 108mM K3PO4(pH~11) * after preparation of the solution above, store it in the fridge at 4C overnight. Prepare a funnel with filter paper and add a teaspoonful active carbon powder, then filter the solution through the powder on the following day. Add glycerol to the filtrate so that its final concentration becomes 2%(v/v). Store it in the fridge at 4C.. Procedures 1. Collect pistil and put them in a plastic tube of the fixative. Leave the tube for at least 2hr at room temperature. 2. Exchange the fixative to 70%EtOH and leave for 10min at RT. After that, do the same treatment using 50, 30%EtOH and DW. 3. Exchange the DW to the ATS carefully. Leave it overnight at room temperature. 4. Exchange ATS to DW carefully because each pistil must be very softened. Leave it for 10 minutes at room temperature. 5. Exchange DW to DABS carefully and leave for at least 2hr under dark condition using a piece of aluminum foil at room temperature. You do not have to wash the specimen after this treatment. 6. Put each pistil with extra DABS on the slide glass, and then put a cover slip on it carefully from the end of pistil with avoiding bubble contamination.. 39.

(50)  Part 2  Dispersal mechanism of Asarum macranthum. 40.

(51) Introduction Seed dispersal is the process that seeds are transported away from their parents. This process is helpful to plant propagation, migration, and expansion. Because of the limited mobility, most plants rely on dispersal agents, including biotic or abiotic agents to disperse seeds. Different behavior, foraging pattern and efficiency of agents strongly influence the population dynamic and distribution. Plants in genus Asarum, usually form a small, restricted population. Convergent dispersal agents coevolved with different seed or fruit morphology. Asarum fruits a capsule which produce 40~60 seeds. Each seed is adhering with a lipid rich aril which is called elaiosome. The lipid-rich appendage or nutrient aril is the common character of ant dispersal syndrome (Berg, 1975; Handel, 1990; Liao and Wu, 2000). Therefore, the corresponding vectors can be speculated from the seed characters. Seed dispersal by ants, termed myrmecochory (myrmēx=ant, kōrē=dispersal), is first investigated in Europe in 1906 (Sernander, 1906), and started in other continents as well (Handel, 1990). Until now, there are more than 80 families, 3000 species around the world are described as myrmecochory (Gomez and Espadaler, 1998; Giladi, 2006). Ants and plants maintain a stable relationship around the world. Both of them benefit from each other. Plants provide elaiosomes to ants. Ants gnaw the myrmecochorous seeds back to their nests, and the elaiosomes are eaten or fed to larva as a nutrient supply (Hanzawa et al, 1988; Whitney, 2002). On the other hand, ant behavior and foraging pattern successfully present an effective dispersal (Handel, 1990). Furthermore, many hypotheses arise about advantages of ant dispersal. Gilada (2006) review these hypothesis, three of them have been widely discussed. First, “directed dispersal” hypothesis- seeds are directly moved to the nest, where the nutrient level is enriched with phosphorous and nitrogen (Culver and Beattie, 1978, 1980). Second, “predator avoidance” hypothesis- with seeds transported to the nest underground, predators are hardly to discover these seeds (Smith et al, 1989). Third,. 41.

(52) “distance dispersal” hypothesis- seeds are moved away from their parents are effectively reduced the parent-offspring competition (Gomez and Espadaler 1998). In conclusion, with the benefits from each other, ants and plants develop a stable relationship. Among the myrmecochorous relationship, two topics will be discussed in this study. First, ants take seeds directly back to their nest. After taking the elaiosome off, abandoned seeds are discarded in the nest and wait for a suitable time to germinate. Therefore, in our hypothesis, distribution of seedlings of A. macranthum should have a strong correlation with ant nest distribution. Second, dispersal distance strongly influences the population size and distribution. While ants move seeds from seed location to ant nest, the dispersal distance is relatively short compared with other vertebrate dispersal systems. Global mean distance of myrmecochory is 96 centimeters long (Gomez and Espadaler, 1998). Short distance dispersal model should influence the population distribution of the plants in genus Asarum. In conclusion, population distributions of plant are influenced by both dispersal distance and agent distribution. Plants in genus Asarum may be an ideal plant to study the seed dispersal phenomenon. Therefore, this study present two aims: (1) Revealing agent composition, behavior, dispersal distance, and elaiosome function to generalize the dispersal background. (2) Surveying the spatial pattern of A.macranthum and ant nest to find out their relationship.. 42.

(53) Material and Methods Dispersal agents observation Fresh and mature seeds were collected the in the field, and several of them are randomly put in study site as baits to attract the seed dispersal agents. Digital video recorder was set to record the whole dispersal process. Totally more than 500 seeds are used in 2009 & 2010. To our preliminary observation, ants are the dispersal agent of A. macranthum. Agent species, moving behavior and foraging model (grouped or solitary) were recorded to reveal the seed dispersal mechanism of A. macranthum. The dispersal agent is defined which could transport seeds more than 5 cm. Animals directly gnawing or eating the seeds without transporting will not be recognized as dispersal agents. All agents are preserved in 70% ethanol in department of Life Science, National Taiwan Normal University, Taipei, Taiwan for further identification.. Measure dispersal distance In order to realize the seed fate after dispersal and the distance that agents carry. I put mature seeds in 100 small depots. Each depot contained three seeds, and was settled randomly in the study site without overlapping continually. From the previously observation, I have already confirmed the major seed dispersal agent are ants. Therefore, when seed were removed, researchers followed the foraging route back to their nest, and measured the linear dispersal distance from source (seed depot) to the sink (ant nest). Any seeds dropped in midway will not be considered. All the data were imputed to the statistic software- JMP to map the frequency distribution of the dispersal distance and to analysis the influence that different ant species contributed to seed dispersal.. Function of elaiosome Surveying the previously image that captured by the digital video recorder, ants. 43.